The enteric nervous system (ENS) is unique because it can mediate reflex activity without involvement of the CNS. The ENS thus contains intrinsic primary afferent neurons and complete neuronal microcircuits. Neither the organization nor the function of the ENS are currently well understood. We have established that serotonin (5-HT) is an enteric neurotransmitter and have developed new methods for the study of enteric microcircuits that have shown that individual myenteric ganglia are not alike. We now plan to continue these studies to characterize the peripheral neural ("M") type of 5-HT receptor, to determine how enteric serotonergic neurons are anatomically and functionally integrated into the ENS, and finally to find the intrinsic enteric primary afferent neurons. Structures that have 5-HT "M" receptors will be identified. Studies of modifications of the molecular structure of 5-HT that affect physiological efficacy and produce specific antagonists will be continued. A photoaffinity probe is being characterized, using radioligand binding assays and intracellular recordings from 5-HT-responsive myenteric neurons. The characterized probe will be employed for molecular identification of the receptor and its high resolution localization. In relating 5-HT to ganglionic heterogeneity, we will determine what is unique about the subset of myenteric ganglia that contain serotonergic neurons (their projections, their inputs [including a possible preferential innervation from the submucosa, and their metabolic activity). Finally, we will test the hypothesis that the intrinsic primary afferents of the ENS are submucosal pseudounipolar neurons that project to the mucosa and myenteric plexus, and receive no synaptic input. Candidate neurons will first be identified and characterized physiologically. They will then be injected intracellularly with markers for LM examination of shape and projections, and EM verification of the absence of synapses. Responsiveness of the cells to 5-HT will be evaluated physiologically and putative transmitters will be examined immunocytochemically. The goal of this project, enhanced understanding of the ENS, is important medically as well as physiologically; malfunction of the ENS causes a great deal of human morbidity, the pathogenesis of which is unclear and the treatment for which is therefore inadequate.